C. antarctica lipase variants

ABSTRACT

A lipase variant of a parent lipase comprising a trypsin-like catalytic triad including an active serine located in a predominantly hydrophobic, elongated binding pocket of the lipase molecule and, located in a critical position of a lipid contact zone of the lipase structure, an amino acid residue different from an aromatic amino acid residue, which amino acid residue interacts with a lipid substrate at or during hydrolysis, in which lipase variant said amino acid residue has been replaced by an aromatic amino acid residue so as to confer to the variant an increased specific activity as compared to that of the parent lipase. The parent lipase may be a C. antarctica lipase A essentially free from other substances from C. antarctica, which comprises the amino acid sequence shown in SEQ ID No. 2, or a variant of said lipase which (1) has lipase activity, (2) reacts with an antibody reactive with at least one epitope of C. antarctica lipase A having th eamino acid sequence SEQ ID No. 2, and/or (3) is encoded by a nucleotide sequence which hybridizes with an oligonucleotide probe prepared on the basis of the full or partial nucleotide sequence shown in SEQ ID No. 1 encoding the C. antarctica lipase A.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a PCT/DK93/00225 filed Jul. 5, 1993, which isincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to novel lipase enzyme variants withimproved properties, DNA constructs coding for the expression of saidvariants, host cells capable of expressing the variants from the DNAconstructs, as well as a method of producing the variants by cultivationof said host cells. Furthermore, the present invention relates to arecombinant essentially pure Candida antarctica lipase and variantsthereof as well as a DNA sequence encoding the said lipase or variantsthereof.

BACKGROUND OF THE INVENTION

A wide variety of lipases of microbial and mammalian origin are known.The amino acid sequence of many of these lipases have been elucidatedand analyzed with respect to structural and functional elementsimportant for their catalytic function, see, for instance, Winkler etal., 1990 and Schrag et al., 1991. It has been found that the lipaseenzyme upon binding of a lipid substrate and activation undergoes aconformational change, which inter alia, results in an exposure of theactive site to the substrate. This conformational change together withthe presumed interaction between enzyme and substrate have beendiscussed by, inter alia, Brady et al., 1990, Brzozowski et al., 1991,Derewenda et al., 1992.

Based on the knowledge of the structure of a number of lipases, it hasbeen possible to construct lipase variants having improved properties byuse of recombinant DNA techniques. Thus, WO 92/05249 discloses theconstruction of certain lipase variants, in which the lipid contact zonehas been modified so as to provide the variants with different substratespecificities and/or an improved accessibility of the active site of thelipase to a lipid substrate. The modifications involve changing theelectrostatic charge, hydrophobicity or the surface conformation of thelipid contact zone by way of amino acid substitutions.

Although the structural and functional relationship of lipases have beenthe subject of a number of studies as described in the above citedreferences, the research has mainly focused on the macroscopiccharacteristics of the lipases upon substrate binding and activation,whereas the identity of the amino acids actually involved in thesubstrate binding and catalytic activity has been discussed only to alesser extent.

SUMMARY OF THE INVENTION

By sequence alignment analysis combined with analysis of the structureand activity of a number of lipases, the present inventors have nowsurprisingly found that the presence of certain amino acids, especiallytryptophan, in a critical position of the lipase seems to be importantfor optimal catalytic activity.

It is consequently an object of the present invention to modify lipaseswhich do not comprise such an amino acid residue in the criticalposition (which lipases in the present context are termed parentlipases) by replacing the amino acid residue located in this positionwith an amino acid residue which gives rise to a variant having anincreased specific activity.

More specifically, in one aspect the present invention relates to alipase variant of a parent lipase comprising a trypsin-like catalytictriad including an active serine located in a predominantly hydrophobic,elongated binding pocket of the lipase molecule and, located in acritical position of a lipid contact zone of the lipase structure, anamino acid residue different from an aromatic amino acid residue, whichinteracts with a lipid substrate at or during hydrolysis, in whichlipase variant said amino acid residue has been replaced by an aromaticamino acid residue so as to confer to the variant an increased specificactivity as compared to that of the parent lipase.

In the present context, the term "trypsin-like" is intended to indicatethat the parent lipase comprises a catalytic triad at the active sitecorresponding to that of trypsin, i.e. the amino acids Ser, His and oneof Asp, Glu, Asn or Gln.

Lipases degrade triglycerides down to fatty acids, glycerol and di-and/or monoglycerides. The lipase action depends on interfacialactivation of the lipase in the presence of substrate surfaces. Onactivation lipases change their conformation in such a manner that theirsurface hydrophobicity in an area around the active site is increased.The interfacial activation of lipases is discussed by Tilbeurgh et al.(1993).

All lipases studied until now have been found to comprise at least onesurface loop structure (also termed a lid or a flap) which covers theactive serine when the lipase is in inactive form (an example of such alipase is described by Brady et al., 1990). When the lipase isactivated, the loop structure is shifted to expose the active siteresidues, creating a surface surrounding the active site Ser, which hasan increased surface hydrophobicity and which interacts with the lipidsubstrate at or during hydrolysis. For the present purpose, this surfaceis termed the "lipid contact zone", intended to include amino acidresidues located within or forming part of this surface, optionally inthe form of loop structures. These residues may participate in lipaseinteraction with the substrate at or during hydrolysis where the lipasehydrolyses triglycerides from the lipid phase when activated by contactwith the lipid surface.

The lipid contact zone contains a binding area (a so-called bindingpocket) for the lipid substrate which is the part of the lipid contactzone to which the lipid substrate binds before hydrolysis. This bindingarea again contains a so-called hydrolysis pocket, which is situatedaround the active site Ser, and in which the hydrolysis of the lipidsubstrate is believed to take place. In all known lipases to date thelipid contact zone is easily recognized, e.g. from a three-dimensionalstructure of the lipase created by suitable computer programs. Theconformation of an inactive and activated lipase, respectively, is shownin FIG. 1 which is further discussed below.

In the present context, the "critical position" of the lipase moleculeis the position in the lipid contact zone of the lipase molecule, whichis occupied by an amino acid residue which interacts with the lipidsubstrate and which is different from an aromatic amino acid residue.

In another aspect the present invention relates an C. antarctica lipaseA which is essentially free from other C. antarctica substances andwhich comprises the amino acid sequence identified in SEQ ID No. 2 or avariant thereof which

1) has lipase activity,

2) reacts with an antibody reactive with at least one epitope of the C.antarctica lipase having the amino acid sequence shown in SEQ ID No. 2,and/or

3) is encoded by a nucleotide sequence which hybridizes with anoligonucleotide probe prepared on the basis of the full or partialnucleotide sequence shown in SEQ ID No. 1 encoding the C. antarcticalipase A.

The C. antarctica lipase A of the invention has a number of desirableproperties including a high thermostability and activity at acidic pHand may advantageously be produced by use of recombinant DNA techniques,e.g. using the procedures described below. Thus, the lipase A of theinvention may be obtained in a higher purity and a higher amount thanthe C. antarctica lipase A purified from wild type C. antarctica whichis described in WO 88/02775.

Furthermore, the present invention relates to a DNA sequence encodingthe C. antarctica lipase A having the amino acid sequence identified inSEQ ID No. 2 or a modification of said DNA sequence encoding a variantof the C. antarctica lipase A as defined above.

In the present context "C. antarctica lipase A" is used inter-changeablywith "lipase A" and the variant of the C. antarctica lipase A is termed"lipase A variant".

The present invention also relates to a DNA construct comprising a DNAsequence encoding a lipase variant as indicated above or a DNA sequenceencoding the C. antarctica lipase A, a recombinant expression vectorcarrying said DNA construct, a cell transformed with the DNA constructor the expression vector, as well as a method of producing a lipasevariant of the invention by culturing said cell under conditionsconducive to the production of the lipase variant, after which thelipase variant is recovered from the culture.

It will be understood that lipase variants of the present inventionhaving an increased specific activity as compared to their parentlipases may be used for the same purposes as their parent lipases,advantageously in a lower amount due to their higher specific activity.

Accordingly, the present invention relates to the use of a lipasevariant of the invention as a detergent enzyme; as a digestive enzyme;in ester hydrolysis, ester synthesis or interesterification; or the useof the lipase variant to avoid pitch trouble arising, e.g., in processesfor preparing mechanical pulp and in paper-making processes usingmechanical pulp.

DETAILED DISCLOSURE OF THE INVENTION

As indicated above, the present inventors have found that the presenceof certain aromatic amino acids, especially tryptophan, located in thelipid contact zone of the lipase molecule is important for optimalcatalytic activity.

The importance of the presence of an aromatic amino acid residue and inparticular a tryptophan residue was found in connection with a study ofmutants of a Humicola lanuginosa lipase which comprises a tryptophanresidue at the critical position in the lipid contact zone, i.e. theamino acid number in the amino acid sequence of the H. lanucinosa lipasepublished in EP 0 305 216. In the H. lanuginosa mutants this istryptophan residue was replaced by phenylalanine, tyrosine, histidine,isoleucine, glutamic acid and glycine, respectively. It was found thatthe specific activity of these mutants decreased (in the order indicatedabove) from 100% of the wild type lipase to about 10% for thephenylalanine mutant and down to about 2% for the glycine mutant.

Without being limited to any theory it is presently believed that theamino acid residue present in the critical position, e.g. on top of orin the proximity of the active serine, may be involved in a)stabilization of the tetrahedral intermediate formed from the lipase andthe substrate during the activation of the lipase, and b) in theactivation of the replacement of the lid region covering the activeserine in the inactive enzyme. When tryptophan is present in thisposition, it is contemplated that optimal performance with respect to a)as well as b) above is obtained. Thus, it is believed that tryptophangives rise to the formation of the most stable tetrahedral intermediate(which means a lowering of the activation energy needed for thecatalysis to take place), and further improves the performance of theenzyme with respect to the activation of the lid opening which isessential for any catalysis to take place.

In connection with a) above it has been observed that the best actinglipase variants contain an unsaturated ring system in the side-chain.The biggest unsaturated system is tryptophan, then tyrosine,phenylalanine and histidine. These sidechains have a pi-electron system("the unsaturation") that could be important for the proton transfer inthe catalysis resulting in a lower activation energy for creating thetetrahedral intermediate where proton transfer has taken place fromactive site histidine to serine to the oxyanion hole created after lidactivation and opening.

From the above theoretical explanation it will be understood that theoptimal amino acid to be present in the critical position, e.g. on topof or in the proximity of the active serine, is tryptophan. However,when the parent lipase is one which does not contain any aromatic aminoacid residue or any amino acid residue with an unsaturated ring systemin the side-chain in this position, such amino acids may advantageouslybe substituted into this position.

Thus, when the parent lipase, in the critical position, has an aminoacid residue which does not comprise an unsaturated ring system in theside-chain, an amino acid residue having such an unsaturatedring-system, e.g. an aromatic amino acid (tryptophan, tyrosine,phenylalanine or histidine) may be substituted into the criticalposition. When the amino acid residue in the critical position of theparent lipase is histidine, it may advantageously be replaced byphenylalanine, tyrosine and most preferably tryptophan, when the aminoacid residue is tyrosine, it may advantageously be replaced byphenylalanine and most preferably tryptophan, and when the amino acidresidue is phenylalanine it may advantageously be replaced bytryptophan.

While the critical position in some lipases is contemplated to be anyposition within the lipid contact zone, the critical position willnormally be located in the binding pocket of the lipase molecule, andpreferably in the hydrolysis pocket thereof. For most lipases it isbelieved that the critical amino acid residue is positioned on top of orin the proximity of the active site.

The amino acid residue occupying this position may be identified in anylipase by 1) sequence alignment studies in which the amino acid sequenceof the lipase in question is aligned with the amino acid sequence ofother lipases, in which the amino acid residue positioned on top of orin the proximity of the active serine has been identified, so as toidentify the presumed position of said amino acid residue, and/or 2) ananalysis of the three-dimensional structure of the lipase in questionusing standard display programmes such as INSIGHT (Biosym TechnologiesInc., San Diego, USA), so as to identify the amino acid sequence on topof or in the proximity of the active serine.

More specifically, on the basis of a computer program such as INSIGHTdisplaying lipase coordinates in accordance with well-known technology,it is simple to point out which part of the lipase contains the lipidcontact zone. 1/ if the structure of the lipase is in a non-activatedform, the lipid contact zone is identified by the direction ofsidechains of the active site Ser. 2/ if the structure is in theactivated form one may additionally base the identification on acolouring of all hydrophobic residues in a colour different from theother residues. By this procedure in which a Corey, Pauling, Koltunmodel of the structure is created, the hydrophobic surface specific forthe lipid contact zone may be identified. The active site Ser is locatedwithin this more hydrophobic part of the molecule.

In some lipases the critical amino acid residue is located in thesurface loop structure covering the active site, or in one or more ofthe surface loop structures found to form part of the surface of thelipid contact zone, such as of the binding pocket or hydrolysis pocket.

Although the critical position is normally considered to be constitutedof only one amino acid residue it may be advantageous to replace two ormore residues, preferably with a tryptophan residue as explained above,in order to obtain a further increased specific activity.

It is contemplated that it is possible to increase the specific activityof parent lipases which do not have a tryptophan residue in the criticalposition at least 2 times, such as at least 3 and preferably at least 4or even 5, 6 or 7 times by modifications as diclosed herein.

It is contemplated that lipase variants as defined herein having anincreased substrate specificity may be prepared on the basis of parentlipases of various origins. Thus, the parent lipase may be a microbiallipase or a mammalian lipase.

When the parent lipase is a microbial lipase, it may be selected fromyeast, e.g. Candida, lipases, bacterial, e.g. Pseudomonas, lipases orfungal, e.g. Humicola or Rhizomucor lipases.

One preferred lipase variant is one, in which the parent lipase isderived from a strain of Candida antarctica, in particular one in whichthe parent lipase is lipase A of C. antarctica, preferably the one whichhas the amino acid sequence shown in SEQ ID No. 2 or a lipase A variantthereof as defined herein. The lipase variant of this C. antarcticalipase A preferably has the amino acid sequence shown in SEQ ID No. 2 inwhich the phenylalanine 139 of the parent lipase has been replaced by atryptophan residue. The construction of this variant and the analysis ofthe properties thereof is discussed in Example 3, 5 and 6.

A lipase variant of the invention may, as mentioned above, be preparedon the basis of a parent lipase derived from a strain of a Pseudomonasspecies, e.g. Ps. fragi. An example of a suitable Ps. frapi lipase whichhas an amino acid residue different from tryptophan positioned on top ofor in the proximity of the active serine, is the one described by Aoyamaet al., 1988. A lipase variant according to the present invention may beconstructed by replacing the phenylalanine residue 29 in the amino acidsequence of said lipase shown in SEQ ID No. 3 by a tryptophan residue.

An example of a fungal lipase suitable as a parent lipase for theconstruction of a lipase variant of the invention is one derived fromRhizopus, especially from R. delemar or R. niveus, the amino acidsequence of which latter is disclosed in, e.g., JP 64-80290. In order toconstruct a lipase variant according to the present invention from thisparent lipase, the alanine residue at position 117 is to be replacedwith an aromatic amino acid residue such as tryptophan. The sequencealignment of the R. niveus lipase sequence (SEQ ID No. 5) and anRhizomucor miehei lipase sequence (containing a tryptophan residue) (SEQID No. 4) is illustrated below. From this alignment the criticalposition of the R. niveus lipase may be determined. ##STR1##

The present inventors have surprisingly found that non-pancreaticlipases such as gastric, lingual, or hepatic lipases have the commonfeature that the amino acid residue which has been identified to be theone located in the critical position of the lipase molecule, normally ontop of or in the proximity of the active serine, is different fromtryptophan. This is in contrast to pancreatic lipases which generallyhave been found to have a tryptophan residue in this position. Thus, inthe present context, non-pancreatic mammalian lipases may advantageouslybe used as "parent lipases" for the construction of lipase variants ofthe invention.

Accordingly, lipase variants as disclosed herein which is of mammalianorigin is advantageously prepared from a parent lipase ofnon-pancreatic, such as gastric, lingual or hepatic origin. Suchmammalian lipases may be derived from humans, rats, mice, pigs, dogs orother mammals. Specific examples of such mammalian lipases includes arat lingual lipase having the sequence identified as A23045 (Docherty etal., 1985), a rat hepatic lipase having the sequence identified asA27442 (Komaromy and Schotz, 1987), a human hepatic lipase having thesequence identified as A33553 (Datta et al., 1988), a human gastriclipase having the sequence identified as S07145 (Bodmer et al., 1987),and a Bio Salt Activated Lipase (BSAL) having the sequence identified asA37916 (Baba et al., 1991) all of which were analysed with respect tothe critical position in the sequence alignment analysis illustratedbelow. The pancreatic lipases included in this sequence alignment studywere a murine pancreatic lipase, A34671 (Grusby et al., 1990), a porcinepancreatic lipase, A00732 (Caro et al., 1981), a human pancreaticlipase, A34494 (Lowe et al., 1989), and a canine pancreatic lipasehaving the sequence B24392 (Mickel et al., 1989). The amino acidsequences of each of the lipases mentioned have the accession numberslisted above and are available from publically available databases.##STR2##

As mentioned above the present invention also relates to a C. antarcticalipase A essentially free from other C. antarctica substances, which hasthe amino acid sequence shown in SEQ ID No. 2 or a variant therof which

1) has lipase activity,

2) reacts with an antibody reactive with at least one epitope of C.antarctica lipase A having the amino acid sequence shown in SEQ ID No.2, and/or

3) is encoded by a nucleotide sequence which hybridizes with anoligonucleotide probe prepared on the basis of the full or partialnucleotide sequence shown in SEQ ID No. 1 encoding the C. antarcticalipase A.

In the present context, the term "variant" is intended to indicate alipase A variant which is derived from the C. antarctica lipase A havingthe amino acid sequence shown in SEQ ID No. 2, or a naturally occurringvariant. Typically, the variant differ from the native lipase A by oneor more amino acid residues, which may have been added or deleted fromeither or both of the N-terminal or C-terminal end of the lipase,inserted or deleted at one or more sites within the amino acid sequenceof the lipase or substituted with one or more amino acid residueswithin, or at either or both ends of the amino acid sequence of thelipase.

Furthermore, the variant of the invention has one or more of thecharacterizing properties 1)-3) mentioned above. Property 1), i.e. the"lipase activity" of the variant may be determined using any knownlipase assay, e.g. the Standard LU assay described in the Methodssection below.

Property 2), i.e. the reactivity of the variant of the invention with anantibody raised against or reactive with at least one epitope of the C.antarctica lipase A having the amino acid sequence shown in SEQ ID No. 2below may be determined by polyclonal antibodies produced in a knownmanner, for instance by immunization of a rabbit with the C. antarcticalipase A of the invention. The antibody reactivity may be determinedusing assays known in the art, examples of which are Western Blotting orradial immunodiffusion assay.

Property 3) above, involving hybridization, may be performed using anoligonucleotide probe prepared on the basis of the full or partial cDNAsequence encoding the C. antarctica lipase A, the amino acid sequence ofwhich is identified in SEQ ID No. 2, as a hybridization probe in ahybridization experiment carried out under standard hybridizationconditions. For instance, such conditions are hybridization underspecified conditions, e.g. involving presoaking in 5×SSC andprehybridizing for 1 h at ˜40° C. in a solution of 20% formamide,5×Denhardt's solution, 50 mM sodium phosphate, pH 6.8, and 50 μg ofdenatured sonicated calf thymus DNA, followed by hybridization in thesame solution supplemented with 100 μM ATP for 18 h at ˜40° C., or othermethods described by e.g. Sambrook et al., 1989.

The nucleotide sequence on the basis of which the oligonucleotide probeis prepared is conveniently the DNA sequence shown in SEQ ID No. 1.

As stated above in a further aspect the present invention relates to aDNA sequence encoding C. antarctica lipase A having the amino acidsequence shown in SEQ ID No. 2 or a modification of said DNA sequencewhich encodes a variant of C. antarctica lipase A which

1) has lipase activity,

2) reacts with an antibody reactive with at least one epitope of the C.antarctica lipase A having the amino acid sequence shown in SEQ ID No.2, and/or

3) is encoded by a nucleotide sequence which hybridizes with anoligonucleotide probe prepared on the basis of the full or partialnucleotide sequence shown in SEQ ID No. 1 encoding the C. antarcticalipase A.

Examples of suitable modifications of the DNA sequence are nucleotidesubstitutions which do not give rise to another amino acid sequence ofthe encoded enzyme, but which may correspond to the codon usage of thehost organism into which the DNA sequence is introduced or nucleotidesubstitutions which do give rise to a different amino acid sequence,without, however, impairing the above stated properties of the enzyme.Other examples of possible modifications are insertion of one or morenucleotides into the sequence, addition of one or more nucleotides ateither end of the sequence and deletion of one or more nucleotides ateither end of or within the sequence.

Methods of Preparing Lipase Variants of the Invention

Several methods for introducing mutations into genes are known in theart. After a brief discussion of cloning lipase-encoding DNA sequences,methods for generating mutations at specific sites within thelipase-encoding sequence will be discussed.

Cloning a DNA Sequence Encoding a Lipase

The DNA sequence encoding a parent lipase or the C. antarctica lipase Aas defined herein may be isolated from any cell or microorganismproducing the lipase in question by various methods, well known in theart. First a genomic DNA and/or cDNA library should be constructed usingchromosomal DNA or messenger RNA from the organism that produces thelipase to be studied. Then, if the amino acid sequence of the lipase isknown, homologous, labelled oligonucleotide probes may be synthesizedand used to identify lipase-encoding clones from a genomic library ofbacterial DNA, or from a fungal cDNA library. Alternatively, a labelledoligonucleotide probe containing sequences homologous to lipase fromanother strain of bacteria or fungus could be used as a probe toidentify lipase-encoding clones, using hybridization and washingconditions of lower stringency.

Yet another method for identifying lipase-producing clones would involveinserting fragments of genomic DNA into an expression vector, such as aplasmid, transforming lipase-negative bacteria with the resultinggenomic DNA library, and then plating the transformed bacteria onto agarcontaining a substrate for lipase. Those bacteria containinglipase-bearing plasmid will produce colonies surrounded by a halo ofclear agar, due to digestion of the substrate by secreted lipase.

Alternatively, the DNA sequence encoding the enzyme may be preparedsynthetically by established standard methods, e.g. the phosphoamiditemethod described by S. L. Beaucage and M. H. Caruthers (1981) or themethod described by Matthes et al. (1984). According to thephosphoamidite method, oligonucleotides are synthesized, e.g. in anautomatic DNA synthesizer, purified, annealed, ligated and cloned inappropriate vectors.

Finally, the DNA sequence may be of mixed genomic and synthetic, mixedsynthetic and cDNA or mixed genomic and cDNA origin prepared by ligatingfragments of synthetic, genomic or cDNA origin (as appropriate), thefragments corresponding to various parts of the entire DNA sequence, inaccordance with standard techniques. The DNA sequence may also beprepared by polymerase chain reaction (PCR) using specific primers, forinstance as described in U.S. Pat. No. 4,683,202 or R. K. Saiki et al.(1988).

Site-Directed Mutagenesis of the Lipase-Encoding Sequence

Once a lipase-encoding DNA sequence has been isolated, and desirablesites for mutation identified, mutations may be introduced usingsynthetic oligonucleotides. These oligonucleotides contain nucleotidesequences flanking the desired mutation sites; mutant nucleotides areinserted during oligonucleotide synthesis. In a specific method, asingle-stranded gap of DNA, bridging the lipase-encoding sequence, iscreated in a vector carrying the lipase gene. Then the syntheticnucleotide, bearing the desired mutation, is annealed to a homologousportion of the single-stranded DNA. The remaining gap is then filled inwith DNA polymerase I (Klenow fragment) and the construct is ligatedusing T4 ligase. A specific example of this method is described inMorinaga et al. (1984). U.S. Pat. No. 4,760,025 discloses theintroduction of oligonucleotides encoding multiple mutations byperforming minor alterations of the cassette, however, an even greatervariety of mutations can be introduced at any one time by the Morinagamethod, because a multitude of oligonucleotides, of various lengths, canbe introduced.

Another method of introducing mutations into lipase-encoding sequencesis described in Nelson and Long (1989). It involves the 3-stepgeneration of a PCR fragment containing the desired mutation introducedby using a chemically synthesized DNA strand as one of the primers inthe PCR reactions. From the PCR-generated fragment, a DNA fragmentcarrying the mutation may be isolated by cleavage with restrictionendonucleases and reinserted into an expression plasmid.

Expression of Lipase Variants

According to the invention, a C. antarctica lipase A-coding sequence ora mutated lipase-coding sequence produced by methods described above orany alternative methods known in the art, can be expressed, in enzymeform, using an expression vector which typically includes controlsequences encoding a promoter, operator, ribosome binding site,translation initiation signal, and, optionally, a repressor gene orvarious activator genes. To permit the secretion of the expressedprotein, nucleotides encoding a "signal sequence" may be inserted priorto the lipase-coding sequence. For expression under the direction ofcontrol sequences, a target gene to be treated according to theinvention is operably linked to the control sequences in the properreading frame. Promoter sequences that can be incorporated into plasmidvectors, and which can support the transcription of the mutant lipasegene, include but are not limited to the prokaryotic β-lactamasepromoter (Villa-Kamaroff et al. (1978) and the tac promoter (DeBoer, etal., 1983). Further references can also be found in "Useful proteinsfrom recombinant bacteria" (1980).

According to one embodiment a strain of Bacillus, e.g. B. subtilis, B.licheniformis or B. lentus, or a strain of E. coli is transformed by anexpression vector carrying the lipase A or the mutated DNA. Ifexpression is to take place in a secreting microorganism such as B.subtilis a signal sequence may follow the translation initiation signaland precede the DNA sequence of interest. The signal sequence acts totransport the expression product to the cell wall where it is cleavedfrom the product upon secretion. The term "control sequences" as definedabove is intended to include a signal sequence, when is present.

The lipase or lipase variants of the invention may further be producedby using a yeast cell has a host cell. Examples of suitable yeast hostcells include a strain of Saccharomyces, such as S. cerevisiae, or astrain of Hansenula, e.g. H. polymorpha or Pichia, e.g. P. pastoris.

In a currently preferred method of producing lipase A or lipase variantsof the invention, a filamentous fungus is used as the host organism. Thefilamentous fungus host organism may conveniently be one which haspreviously been used as a host for producing recombinant proteins, e.g.a strain of Aspergillus sp., such as A. niger, A. nidulans or A. oryzae.The use of A. oryzae in the production of recombinant proteins isextensively described in, e.g. EP 238 023.

For expression of lipase variants in Aspergillus, the DNA sequencecoding for the lipase A or the lipase variant is preceded by a promoter.The promoter may be any DNA sequence exhibiting a strong transcriptionalactivity in Aspergillus and may be derived from a gene encoding anextracellular or intracellular protein such as an amylase, aglucoamylase, a protease, a lipase, a cellulase or a glycolytic enzyme.

Examples of suitable promoters are those derived from the genes encodingA. oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, A. nigerneutral α-amylase, A. niger acid stable α-amylase, A. nigerglucoamylase, Rhizomucor miehei lipase, A. oryzae alkaline protease orA. oryzae triose phosphate isomerase.

In particular when the host organism is A. oryzae, a preferred promoterfor use in the process of the present invention is the A. oryzae TAKAamylase promoter as it exhibits a strong transcriptional activity in A.oryzae. The sequence of the TAKA amylase promoter appears in EP 238 023.

Termination and polyadenylation sequences may suitably be derived fromthe same sources as the promoter.

The techniques used to transform a fungal host cell may suitably be asdescribed in EP 238 023.

To ensure secretion of the lipase A or the lipase variant from the hostcell, the DNA sequence encoding the lipase variant may be preceded by asignal sequence which may be a naturally occurring signal sequence or afunctional part thereof or a synthetic sequence providing secretion ofthe protein from the cell. In particular, the signal sequence may bederived from a gene encoding an Aspergillus sp. amylase or glucoamylase,a gene encoding a Rhizomucor miehei lipase or protease, or a geneencoding a Humicola cellulase, xylanase or lipase. The signal sequenceis preferably derived from the gene encoding A. oryzae TAKA amylase, A.niger neutral α-amylase, A. niger acid-stable α-amylase or A. nigerglucoamylase.

The medium used to culture the transformed host cells may be anyconventional medium suitable for culturing Aspergillus cells. Thetransformants are usually stable and may be cultured in the absence ofselection pressure. However, if the transformants are found to beunstable, a selection marker introduced into the cells may be used forselection.

The mature lipase protein secreted from the host cells may convenientlybe recovered from the culture medium by well-known procedures includingseparating the cells from the medium by centrifugation or filtration,and precipitating proteinaceous components of the medium by means of asalt such as ammonium sulphate, followed by chromatographic proceduressuch as ion exchange chromatography, affinity chromatography, or thelike.

It will be understood that the lipase variants of the invention arecontemplated to be active towards the same type of substrates as theirparent lipases, with an improved specific activity. Thus, the lipasevariants of the invention are contemplated to be useful for the samepurposes as their parent lipases.

Accordingly, lipase variants of the invention prepared from a parentlipase useful as a detergent enzyme may be used as an active ingredientin a detergent additive or a detergent composition.

Another contemplated use of lipase variants of the invention, is asdigestive enzymes, e.g. in the treatment of cystic fibrosis.

A third use of the lipase variants of the invention, especially variantsof C. antarctica lipases are in lipase-catalysed processes such as inester hydrolysis, ester synthesis and interesterification. The use oflipases in these processes is discussed in detail in WO 88/02775 (NovoNordisk A/S), the content of which is incorporated herein by reference.Furthermore, as the C. antarctica is a nonspecific lipase, it may beused for randomization, e.g. in the preparation of margarine. Also thelipase variants of the invention may be used to avoid pitch trouble thatarises in the production process for mechanical pulp or in apaper-making process using mechanical pulp, e.g. as described inPCT/DK92/00025 (Novo Nordisk A/S), the content of which is incorporatedherein by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention is described in the following with reference tothe appended drawings, in which

FIG. 1 is a computer model showing the three-dimensional structure ofthe lipid contact zone of the H. lanuginosa lipase described in WO92/05249 when the lipase is in inactive (closed) and active (open) form,respectively. "White" residues represent hydrophobic amino acids (Ala,Val, Leu, Ile, Pro, Phe, Trp, Gly and Met), "yellow" residues representhydrophilic amino acids (Thr, Ser, Gln, Asn, Tyr and Cys), "blue"residues represent positively charged amino acids (Lys, Arg and His),and "red" residues represent negatively charged amino acids (Glu andAsp).

FIGS. 2 and 3 illustrate the scheme for the construction of theexpression plasmid pMT1229 (see Example 1).

The present invention is further illustrated in the following exampleswhich are not intended, in any way, to limit the scope of the inventionas claimed.

MATERIALS

Plasmids and Microorganisms

pBoel777 (p777) (described in EP 0 489 718)

p775 (the construction of which is described in EP 0 238 023)

pIC19H (Marsh et al., Gene 32 (1984), pp. 481-485)

pToC90 (described in WO 91/17243)

Aspergillus oryzae A1560: IFO 4177

E. coli MT172 (a K12 restriction deficient E. coli MC1000 derivative)

GENERAL METHODS

Site-Directed in Vitro Mutagenesis of Lipase Genes

The three different approaches described in WO 92/05249 may be used forintroducing mutations into the lipase genes, i.e. the oligonucleotidesite-directed mutagenesis which is described by Zoller & Smith, DNA,Vol. 3, No. 6, 479-488 (1984), the PCR method as described in Nelson &Long, Analytical Biochemistry, 180, 147-151 (1989), and the so-called"cassette mutagenesis" technique, in which a segment between tworestriction sites of the lipase-encoding region is replaced by asynthetic DNA fragment carrying the desired mutation. Use of the lattertechnique is illustrated in Example 2.

Determination of Lipase Specific Activity

Lipase activity was assayed using glycerine tributyrat as a substrateand gum-arabic as an emulsifier. 1 LU (Lipase Unit) is the amount ofenzyme which liberates 1 μmol titratable butyric acid per minute at 30°C., pH 7.0. The lipase activity was assayed by pH-stat using Radiometertitrator VIT90, Radiometer, Copenhagen. Further details of the assay aregiven in Novo Analytical Method AF 95/5, available on request.

EXAMPLES Example 1

Cloning of Candida antarctica Lipase A

Chromosomal DNA of the C. antarctica strain LF058 (=DSM 3855 depositedwith the Deutsche Sammlung von Mikroorganismen (DSM) on Sep. 29, 1986under the terms of the Budapest Treaty, and further described in WO88/02775) was prepared by opening of frozen cells by grinding withquartz and subsequent extraction of DNA essentially as described byYelton et al., (1984). The purified DNA was cut partially with Sau3Aand, after agarose gel electrophoresis, fragments in the range of 3-9 kbwere isolated. The sized Sau3A fragments were ligated into a BamH1-cut,dephosphorylated plasmid pBR322 (New England Biolabs). The ligation mixwas transformed into the E. coli MT172. Approximately 50,000transformant E. coli colonies were obtained, 80% of which contained aninsert of LF058 DNA.

Using standard colony hybridization techniques (Maniatis et al., 1982)the colonies were screened with the 32^(p) -phosphorylatedoligonucleotide probe NOR 440 (SEQ ID No. 7). NOR 440 is a degenerated(64) 17 mer based on the N-terminal determined from mature C. antarcticalipase A (SEQ ID No. 2). 34 colonies appeared positive after wash at lowstringency (41° C. and 6×SSC). Plasmids were prepared from thesecolonies and Southern analyzed after restriction with BstNl. The probefor the Southern was either the NOR 440 probe (SEQ ID No. 7) used forthe colony hybridization (see above) or a 32^(p) -labelled probe NOR 438(SEQ ID No. 6). NOR 438 is an oligonucleotide (a guess mer) where, at 13positions, a base has been chosen on the basis of codon use in yeastsand filamentous fungi. ##STR3## guess positions indicated

Only one plasmid, pMT1076, contained a band which hybridized both to NOR440 at low stringency (see above) and to NOR 438 at a somewhat higherstringency (55° C. and 1×SSC).

PMT1076 was restriction mapped and the DNA sequence determined by theMaxam-Gilbert method. The sequence covering the open reading frame isshown in SEQ ID No. 1. The open reading frame is seen to encode aputative signal sequence of 21 amino acids (according to the von Heinerules (von Heijne, G. (1986)) and furthermore a propeptide of 10 aminoacids preceding the N-terminal of the mature lipase. The last two aminoacids of the propeptide are Arg Arg, i.e. a typical cleavage site forendoproteolytic processing by enzymes of the S. cereviciae KEX-2 type.The amino acid composition of the mature protein (starting at position32) encoded by the DNA sequence is in agreement with the amino acidcomposition determined for C. antarctica lipase A, cf. the followingtable:

                  TABLE I                                                         ______________________________________                                        Amino acid composition of C. antartica lipase A (CALIP)                                   Deduced from DNA                                                                           By amino acid                                          sequence analysis (MC)                                                      ______________________________________                                        Ala         50           47                                                     Arg 9 9                                                                       Asp/AsN 35 36                                                                 Cys 4 4                                                                       Gln/GlN 35 36                                                                 Gly 28 31                                                                     His 6 6                                                                       Ile 26 24                                                                     Leu 29 30                                                                     Lys 17 17                                                                     Met 2 3                                                                       Phe 20 19                                                                     Pro 33 33                                                                     Ser 26 27                                                                     Thr 27 28                                                                     Trp 5 4                                                                       Tyr 18 16                                                                     Val 27 26                                                                   ______________________________________                                    

Through a number of standard plasmid manipulations (Maniatis et al.,1982) illustrated in FIGS. 2 and 3, the open reading frame of C.antarctica lipase A was placed in the correct orientation between thealpha-amylase promoter of A. oryzae and the glucoamylase transcriptionterminator of A. niger. The resulting expression plasmid pMT1229 wastransformed into A. oryzae A1560 as described in EP 305,216.Transformants were isolated and grown as described in the above citedpatents and the culture supernatants were analyzed for the presence ofC. antarctica lipase A.

Example 2

Construction of a Plasmid Expressing the F135W Variant of Candidaantarctica Lipase A

A 246 bp BamHI/BssHII fragment was synthesized in vitro on the basis ofthe nucleotide sequence of pMT1229 using oligonucleotide primers 3116and 3117 in a PCR reaction. The primer 3117 includes a BssHIIrestriction site and a mutation in the 135 phe codon (TTC) to trp codon(TGG) which is marked with stars.

Oligonucleotide primer 3116 (F135W:256-276) (SEQ ID No. 8) 5'-CAG AACGAG GCG GTG GCC GAC-3'

Oligonucleotide primer 3117 (F135W:566-487) (SEQ ID No. 9) 5'-TTC TTGAGC GCG CGG ATG CCG TCG AGG ATA GCC ATG CCC TCT TCG TAG CCA GCG ATG AAGGCG GCT TTC* C*AG CCT TCG TG-3'

The PCR reaction was performed by mixing the following components andincubating the mixture in a HYBAID™ thermal reactor.

    ______________________________________                                        Template pMT1229                                                                              10     ng/μl  1    μl                                     H.sub.2 O   46.5 μl                                                        10 × PCR buffer   10 μl                                              2 mM dATP   10 μl                                                          2 mM dTTP   10 μl                                                          2 mM dCTP   10 μl                                                          2 mM dGTP   10 μl                                                          primer 3116 50.5 pmol/μl 1 μl                                           primer 3117 70.5 pmol/μl 1 μl                                           Taq polymerase   0.5 μl                                                    Parafin oil   50 μl                                                      Step I    94° C.                                                                           2      min.    1    cycle                                   Step II 94° C. 30 sec.                                                  50° C. 30 sec. 30 cycle                                                72° C. 2 min.                                                         Step III 72° C. 5 min. 1 cycle                                       ______________________________________                                    

The resulting 310 bp fragment was isolated from a 2% agarose gel afterelectrophoresis and digested with BamHI and BssHII restriction enzymes.The resulting 264 bp BamHI/BssHII fragment was likewise isolated from 2%agarose gel. This fragment was then ligated with

    ______________________________________                                        pMT1229        BamHI/XbaI    0.3 kb                                             pMT1229 BssHII/SphI 0.5 kb                                                    pMT1229 SphI/XbaI 5.0 kb                                                    ______________________________________                                    

The ligated DNA was transformed into E. coli strain MT172. Transformantswhich contained correct inserts were selected and their DNA sequence wasdetermined by use of Sequenase (United States Biochemical Corporation).One resulting plasmid (pME-1178) contained a mutation in the amino acidposition 135 (phe was mutated to trp).

pME1178 was cotransformed with pToC90 which included the amdS gene fromA. nidulans as a selective marker into the A. oryzae A1560 strain usingthe procedure described in WO 91/17243. A. oryzae transformants werereisolated twice on selective plates and stable transformants werecharacterized by rocket immunoelectrophoresis, using anti-Candida lipaseA antibody. Candida lipase A produced by a transformant (strain MEA65)was further analyzed for specific activity.

Example 3

Construction of a Plasmid Expressing the F139W Variant of Candidaantarctica Lipase A

A 246 bp BamHI/BssHII fragment was synthesized in vitro on the basis ofthe nucleotide sequence of the plasmid pMT1229 using oligonucleotideprimers 3116 and 3826 in a PCR reaction. The primer 3826 includes aBssHII restriction site and a mutation in the 139 phe codon (TTC) to trpcodon (TGG) which is marked with stars.

Oligonucleotide primer 3116 is shown in Example 2.

Oligonucleotide primer 3826 (F139W:566-487) (SEQ ID No. 10) 5'-TTC TTGAGC GCG CGG ATG CCG TCG AGG ATA GCC ATG CCC TCT TCG TAG CCA GCG ATC*C*AG GCG GCT TTG AAG CCT TCG TG-3'

A PCR reaction was performed by the method described in Example 2. The310 bp fragment was isolated from 2% agarose gel after electrophoresisand digested by BamHI and BssHII restriction enzymes. The resulting 264bp BamHI/BssHII fragment was likewise isolated from 2% agarose gel. Thisfragment was then ligated with

    ______________________________________                                        pMT1229        BamHI/XbaI    0.3 kb                                             pMT1229 BssHII/SphI 0.5 kb                                                    pMT1229 SphI/XbaI 5.0 kb                                                    ______________________________________                                    

The ligated DNA was transformed into E. coli strain MT172. Transformantswhich contained correct inserts were selected and their DNA sequence wasdetermined by use of Sequenase (United States Biochemical Corporation).One resulting plasmid (pME-1229) contained a mutation in the amino acidposition 139 (phe was mutated to trp).

pME1229 was cotransformed with pToC90 which included the amdS gene fromA. nidulans as a selective marker into A. oryzae A 1560 strain. A oryzaetransformants were reisolated twice on selective plates and enzymeactivity of a stable transformant (MEA155) was analyzed by usingtributylene as a substrate as described in Example 5.

Example 4

Construction of a Plasmid Expressing the F135W/F139W Variant of Candidaantarctica Lipase A

A 246 bp BamHI/BssHII fragment was synthesized in vitro usingoligonucleotide primers 3116 and 4224 by a PCR reaction. The primer 4224includes a BssHII restriction site and mutations in the 135 and 139codons (TTC) to trp codons (TGG) which are marked with stars.

The oligonucleotide primer 3116 is shown in Example 2.

Oligonucleotide primer 4224 (F135W:566-487) (SEQ ID No. 11) 5'-TTC TTGAGC GCG CGG ATG CCG TCG AGG ATA GCC ATG CCC TCT TCG TAG CCA GCG ATC*C*AG GCG GCT TTC* C*AG CCT TCG TG-3'

PCR reaction was performed by using the method shown in Example 2. The310 bp fragment was isolated from a 2% agarose gel after electrophoresisand digested with BamHI and BssHII restriction enzymes. The resulting264 bp BamHI/BssHII fragment was likewise isolated from a 2% agarosegel. This fragment was then ligated with

    ______________________________________                                        pMT1229        BamHI/XbaI    0.3 kb                                             pMT1229 BssHII/SphI 0.5 kb                                                    pMT1229 SphI/XbaI 5.0 kb                                                    ______________________________________                                    

The ligated DNA was transformed into E. coli MT172. Transformants whichcontained inserts were selected and their DNA sequence was determined byuse of Sequenase. One resulting plasmid (pME1230) contained twomutations in the amino acid positions 135 and 139 (phe was mutated totrp).

pME1230 was cotransformed with pToC90 which included the amds gene fromA. nidulans as a selective marker into A. oryzae A 1560 strain. A.oryzae transformants were reisolated twice on selective plates andenzyme activity of stable transformants were analyzed by usingtributylene as a substrate as described in Example 5.

Example 5

Purification of C. antarctica Lipase A Variants P139W and F135W/F139Wand Comparison of Specific Activity with their Parent Wild Type C.antarctica Lipase A

The lipase variants and the parent lipase produced as described inExamples 3, 4 and 1, respectively, were purified using the following 4step standard purification procedure.

Step 1: The fermentation broth containing the lipase and lipase variant,respectively, obtained by culturing the transformed A. oryzae cellsdescribed in Examples 1 and 3 above, was centrifuged, and thesupernatant was adjusted to pH 7. Ionic strength was adjusted to 2 mSi.DEAE-Sephadex A-50 (Pharmacia) gel was swollen and equilibrated in 25 mMTris acetate buffer pH 7. The fermentation supernatant was passedthrough DEAE-Sephadex A-50 on scintered glass funnel. The effluentcontaining lipase activity was collected and adjusted to 0.8 M ammoniumacetate.

Step 2: An appropriate column was packed with TSK gel Butyl-Toyopearl650 C and equilibrated with 0.8 M ammonium acetate. The effluentcontaining lipase activity was applied on the column. The bound materialwas eluted with water.

Step 3: The lipase-containing eluate was then applied on aHighperformance Q-Sepharose column. Lipase activity was collected aseffluent. The lipases purified by this method were concentrated to anOptical Density of 1 at 280 nm.

The purity of the lipases was checked by SDS-PAGE showing one band withan molecular weight of about 45 kD. The lipase activity was determinedby use of the method outlined above in the section "General methods".

The lipase activity of the parent wild type lipase was 300 LU/OD₂₈₀ ascompared to 1200 LU/OD₂₈₀ for the lipase variant F139W. On the basis ofOD₂₈₀ absorption without correction for the inserted tryptophan, thespecific activity of the mutant was at least four times higher with theassay used. The lipase activity of the lipase variant F135W/F139W was1400 LU/OD₂₈₀ (without correction for the two additional tryptophans).

Example 6

Thermostability of Candida antarctica Lipase A and the Mutant F139Wthereof

The thermostability of the C. antarctica lipase A and the C. antarcticalipase A variant, was examined by Differential Scanning Calorimetry(DSC) at different pH values. Using this technique, the thermaldenaturation temperature, T_(d), is determined by heating an enzymesolution at a constant programmed rate.

More specifically, the Differential Scanning Calorimeter, MC-2D, fromMicroCal Inc. was used for the investigations. Enzyme solutions wereprepared in 50 mM buffer solutions, cf. the tables below. The enzymeconcentration ranged between 0.6 and 0.9 mg/ml, and a total volume ofabout 1.2 ml was used for each experiment. All samples were heated from25° C. to 90° C. at a scan rate of 90° C./hr.

The results obtained from the analysis is shown in the table below:

    ______________________________________                                        pH       Buffer (50 mM)                                                                            Denaturation temperature.sup.1)                          ______________________________________                                        C. ant. lipase A (WT)                                                           4.5        Acetate     96° C.                                          5 Acetate 95° C.                                                       7 TRIS 93° C.                                                        C. ant. lipase A mutant (F139W)                                                 5          Acetate     84° C.                                          7 TRIS 82° C.                                                        ______________________________________                                         .sup.1) Temperature, at which approximately half the enzyme molecules         present have been denatured thermally during heating                     

The above results show that the pH-optimum for the thermostability of C.antarctica lipase A and the F139W variant is unusually low and that bothenzymes are very thermostable below pH 7. Within the investigated rangethe thermostability of both the Wild Type and the mutant F139W continuesto increase as pH is lowered. This makes both lipases very well suitedfor hydrolysis/synthesis at unusually high temperatures at relativelylow pH values.

References Cited in the Application

Winkler, F. K. et al., (1990), Structure of Human Pancreatic Lipase.Nature, vol. 343, 771-774,

Schrag, J. D. et al., (1991), Ser-His-Glu triad Forms the Catalytic Siteof the Lipase from Geotrichum candidum. Nature, vol. 351, 761-764,

Brady, Leo et al., (1990), A Serine Protease Triad Forms the CatalyticCentre of a Triacylglycerol Lipase. Nature, vol 343, 767-770,

Brzozowski, A. M. et al., (1991), A Model for Interfacial Activation inLipases from the Structure of a Fungal Lipase-inhibitor Complex. Nature,vol. 351, 491-494,

Derewenda, Urszula et al., (1992), Catalysis at the Interface: TheAnatomy of a Conformational Change in a Triglyceride Lipase. Biochem.,31, 1532-1541,

Tilbeurgh et al., Nature, Vol. 362, 814-820, (1993)

Komaromy, M. C. et al., (1987), Cloning of Rat Hepatic Lipase cDNA:Evidence for a Lipase Gene Family. Proc.Natl.Acad.Sci., 84, 1526-1530,

Datta, S. et al., (1988) Human Hepatic Lipase. J.Biol.Chem., 263,1107-1110,

Bodmer, M. W. et al., (1987) Molecular Cloning of a Human Gastric Lipaseand Expression of the Enzyme in Yeast. Biochimica et Biophysica Acta,909, 237-244.

Baba, T. et al., (1991), Structure of Human Milk Bile Salt ActivatedLipase. Biochemistry, 30, 500-510,

Grusby, M. J. et al., (1990), Cloning of an Interleukin-4 Inducible Genefrom Cytotoxic T Lymphocytes and its Identification as a lipase. Cell60, 451-459,

Caro, J. De. et al., (1981) Porcine Pancreatic Lipase. Completion of thePrimary Structure. Biochim.Biophys. Acta 671, 129-138,

Lowe, M. E. et al., (1989), Cloning and Characterization of HumanPancreatic Lipase cDNA. J.Biol.Chem., 264, 20042-20048,

Mickel, F. S. et al., (1989), Structure of the Canine Pancreatic LipaseGene. J.Biol.Chem., 264, 12895-12901,

S. L. Beaucage and M. H. Caruthers, Tetrahedron Letters 22, 1981, pp.1859-1869,

Matthes et al., The EMBO J. 3, 1984, pp. 801-805

R. K. Saiki et al., Science 29, 1988, pp. 487-491

Morinaga et al., 1984, Biotechnology 2: 646-639

Nelson and Long, Analytical Biochemistry 180, 1989, pp. 147-151

Villa-Kamaroff, et al., 1978, Proc. Natl. Acad. Sci. U.S.A. 75:3727-3731)

DeBoer, et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80: 21-25

"Useful proteins from recombinant bacteria" in Scientific American,1980, 242: 74-94.

Marsh et al., Gene 32 (1984), pp. 481-485)

Docherty, A. J. P. et al., (1985), Molecular Cloning and NucleotideSequence of Rat Lingual Lipase cDNA. Nucleic Acids Research, 13,1891-1903,

Maniatis, T. et al., Molecular Cloning, Cold Spring Harbor, 1982,

Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., ColdSpring Harbor, 1989,

Zoller & Smith, DNA, Vol. 3, No. 6, 479-488 (1984),

Nelson & Long, Analytical Biochemistry, 180, 147-151 (1989)

von Heijne, G. Nucl. Acid. Res. 14 (1986), pp. 4683-90,

Yelton et al., PNAS 81 (1984), pp. 1470-74,

Aoyama, S. et al., (1988), Cloning, sequencing and expression of thelipase gen from Pseudomonas fragi IFO-12049 in E. coli. FEBS Lett., 242,36-40,

Derewenda, Zygmunt S. et al., (1992), Relationships Among SerineHydrolases: Evidence for a Common Structural Motif in TriacylglycerideLipases and Esterases,

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 11                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1329 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:1:                        - - GCGGCTCCGG CGGCCGAGAC GCTGGACCGA CGGGCGGCGC TGCCCAACCC CT -            #ACGACGAT     60                                                                 - - CCCTTCTACA CGACGCCATC CAACATCGGC ACGTTTGCCA AGGGCCAGGT GA -            #TCCAATCT    120                                                                 - - CGCAAGGTGC CCACGGACAT CGGCAACGCC AACAACGCTG CGTCGTTCCA GC -            #TGCAGTAC    180                                                                 - - CGCACCACCA ATACGCAGAA CGAGGCGGTG GCCGACGTGG CCACCGTGTG GA -            #TCCCGGCC    240                                                                 - - AAGCCCGCTT CGCCGCCCAA GATCTTTTCG TACCAGGTCT ACGAGGATGC CA -            #CGGCGCTC    300                                                                 - - GACTGTGCTC CGAGCTACAG CTACCTCACT GGATTGGACC AGCCGAACAA GG -            #TGACGGCG    360                                                                 - - GTGCTCGACA CGCCCATCAT CATCGGCTGG GCGCTGCAGC AGGGCTACTA CG -            #TCGTCTCG    420                                                                 - - TCCGACCACG AAGGCTTCAA AGCCGCCTTC ATCGCTGGCT ACGAAGAGGG CA -            #TGGCTATC    480                                                                 - - CTCGACGGCA TCCGCGCGCT CAAGAACTAC CAGAACCTGC CATCCGACAG CA -            #AGGTCGCT    540                                                                 - - CTTGAGGGCT ACAGTGGCGG AGCTCACGCC ACCGTGTGGG CGACTTCGCT TG -            #CTGAATCG    600                                                                 - - TACGCGCCCG AGCTCAACAT TGTCGGTGCT TCGCACGGCG GCACGCCCGT GA -            #GCGCCAAG    660                                                                 - - GACACCTTTA CATTCCTCAA CGGCGGACCC TTCGCCGGCT TTGCCCTGGC GG -            #GTGTTTCG    720                                                                 - - GGTCTCTCGC TCGCTCATCC TGATATGGAG AGCTTCATTG AGGCCCGATT GA -            #ACGCCAAG    780                                                                 - - GGTCAGCGGA CGCTCAAGCA GATCCGCGGC CGTGGCTTCT GCCTGCCGCA GG -            #TGGTGTTG    840                                                                 - - ACCTACCCCT TCCTCAACGT CTTCTCGCTG GTCAACGACA CGAACCTGCT GA -            #ATGAGGCG    900                                                                 - - CCGATCGCTA GCATCCTCAA GCAGGAGACT GTGGTCCAGG CCGAAGCGAG CT -            #ACACGGTA    960                                                                 - - TCGGTGCCCA AGTTCCCGCG CTTCATCTGG CATGCGATCC CCGACGAGAT CG -            #TGCCGTAC   1020                                                                 - - CAGCCTGCGG CTACCTACGT CAAGGAGCAA TGTGCCAAGG GCGCCAACAT CA -            #ATTTTTCG   1080                                                                 - - CCCTACCCGA TCGCCGAGCA CCTCACCGCC GAGATCTTTG GTCTGGTGCC TA -            #GCCTGTGG   1140                                                                 - - TTTATCAAGC AAGCCTTCGA CGGCACCACA CCCAAGGTGA TCTGCGGCAC TC -            #CCATCCCT   1200                                                                 - - GCTATCGCTG GCATCACCAC GCCCTCGGCG GACCAAGTGC TGGGTTCGGA CC -            #TGGCCAAC   1260                                                                 - - CAGCTGCGCA GCCTCGACGG CAAGCAGAGT GCGTTCGGCA AGCCCTTTGG CC -            #CCATCACA   1320                                                                 - - CCACCTTAG                - #                  - #                      - #       1329                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO: 2:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 463 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO: 2:                       - -      Met Arg Val Ser Leu Arg Ser Ile - # Thr Ser Leu Leu Ala Ala        Ala Thr                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Ala Ala Val Leu Ala Ala Pro Ala - # Ala Glu Thr Leu Asp Arg       Arg Ala                                                                                          20 - #                 25 - #                 30             - -      Ala Leu Pro Asn Pro Tyr Asp Asp - # Pro Phe Tyr Thr Thr Pro        Ser Asn                                                                                      35     - #             40     - #             45                  - -      Ile Gly Thr Phe Ala Lys Gly Gln - # Val Ile Gln Ser Arg Lys       Val Pro                                                                                  50         - #         55         - #         60                      - -      Thr Asp Ile Gly Asn Ala Asn Asn - # Ala Ala Ser Phe Gln Leu       Gln Tyr                                                                              65             - #     70             - #     75             - #         80                                                                            - -      Arg Thr Thr Asn Thr Gln Asn Glu - # Ala Val Ala Asp Val Ala        Thr Val                                                                                           - #   85               - #   90               - #         95                                                                               - -      Trp Ile Pro Ala Lys Pro Ala Ser - # Pro Pro Lys Ile Phe Ser       Tyr Gln                                                                                          100 - #                105 - #                110            - -      Val Tyr Glu Asp Ala Thr Ala Leu - # Asp Cys Ala Pro Ser Tyr        Ser Tyr                                                                                      115     - #            120     - #            125                 - -      Leu Thr Gly Leu Asp Gln Pro Asn - # Lys Val Thr Ala Val Leu       Asp Thr                                                                                  130         - #        135         - #        140                     - -      Pro Ile Ile Ile Gly Trp Ala Leu - # Gln Gln Gly Tyr Tyr Val       Val Ser                                                                              145             - #    150             - #    155             - #        160                                                                           - -      Ser Asp His Glu Gly Phe Lys Ala - # Ala Phe Ile Ala Gly Tyr        Glu Glu                                                                                           - #   165              - #   170              - #         175                                                                              - -      Gly Met Ala Ile Leu Asp Gly Ile - # Arg Ala Leu Lys Asn Tyr       Gln Asn                                                                                          180 - #                185 - #                190            - -      Leu Pro Ser Asp Ser Lys Val Ala - # Leu Glu Gly Tyr Ser Gly        Gly Ala                                                                                      195     - #            200     - #            205                 - -      His Ala Thr Val Trp Ala Thr Ser - # Leu Ala Glu Ser Tyr Ala       Pro Glu                                                                                  210         - #        215         - #        220                     - -      Leu Asn Ile Val Gly Ala Ser His - # Gly Gly Thr Pro Val Ser       Ala Lys                                                                              225             - #    230             - #    235             - #        240                                                                           - -      Asp Thr Phe Thr Phe Leu Asn Gly - # Gly Pro Phe Ala Gly Phe        Ala Leu                                                                                           - #   245              - #   250              - #         255                                                                              - -      Ala Gly Val Ser Gly Leu Ser Leu - # Ala His Pro Asp Met Glu       Ser Phe                                                                                          260 - #                265 - #                270            - -      Ile Glu Ala Arg Leu Asn Ala Lys - # Gly Gln Arg Thr Leu Lys        Gln Ile                                                                                      275     - #            280     - #            285                 - -      Arg Gly Arg Gly Phe Cys Leu Pro - # Gln Val Val Leu Thr Tyr       Pro Phe                                                                                  290         - #        295         - #        300                     - -      Leu Asn Val Phe Ser Leu Val Asn - # Asp Thr Asn Leu Leu Asn       Glu Ala                                                                              305             - #    310             - #    315             - #        320                                                                           - -      Pro Ile Ala Ser Ile Leu Lys Gln - # Glu Thr Val Val Gln Ala        Glu Ala                                                                                           - #   325              - #   330              - #         335                                                                              - -      Ser Tyr Thr Val Ser Val Pro Lys - # Phe Pro Arg Phe Ile Trp       His Ala                                                                                          340 - #                345 - #                350            - -      Ile Pro Asp Glu Ile Val Pro Tyr - # Gln Pro Ala Ala Thr Tyr        Val Lys                                                                                      355     - #            360     - #            365                 - -      Glu Gln Cys Ala Lys Gly Ala Asn - # Ile Asn Phe Ser Pro Tyr       Pro Ile                                                                                  370         - #        375         - #        380                     - -      Ala Glu His Leu Thr Ala Glu Ile - # Phe Gly Leu Val Pro Ser       Leu Trp                                                                              385             - #    390             - #    395             - #        400                                                                           - -      Phe Ile Lys Gln Ala Phe Asp Gly - # Thr Thr Pro Lys Val Ile        Cys Gly                                                                                           - #   405              - #   410              - #         415                                                                              - -      Thr Pro Ile Pro Ala Ile Ala Gly - # Ile Thr Thr Pro Ser Ala       Asp Gln                                                                                          420 - #                425 - #                430            - -      Val Leu Gly Ser Asp Leu Ala Asn - # Gln Leu Arg Ser Leu Asp        Gly Lys                                                                                      435     - #            440     - #            445                 - -      Gln Ser Ala Phe Gly Lys Pro Phe - # Gly Pro Ile Thr Pro Pro       Glx                                                                                      450         - #        455         - #        460                     - -  - - (2) INFORMATION FOR SEQ ID NO: 3:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 277 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO: 3:                       - -      Met Asp Asp Ser Val Asn Thr Arg - # Tyr Pro Ile Leu Leu Val       His Gly                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Leu Phe Gly Phe Asp Arg Ile Gly - # Ser His His Tyr Phe His       Gly Ile                                                                                          20 - #                 25 - #                 30             - -      Lys Gln Ala Leu Asn Glu Cys Gly - # Ala Ser Val Phe Val Pro        Ile Ile                                                                                      35     - #             40     - #             45                  - -      Ser Ala Ala Asn Asp Asn Glu Ala - # Arg Gly Asp Gln Leu Leu       Lys Gln                                                                                  50         - #         55         - #         60                      - -      Ile His Asn Leu Arg Arg Gln Val - # Gly Ala Gln Arg Val Asn       Leu Ile                                                                              65             - #     70             - #     75             - #         80                                                                            - -      Gly His Ser Gln Gly Ala Leu Thr - # Ala Arg Tyr Val Ala Ala        Ile Ala                                                                                           - #   85               - #   90               - #         95                                                                               - -      Pro Glu Leu Ile Ala Ser Val Thr - # Ser Val Ser Gly Pro Asn       His Gly                                                                                          100 - #                105 - #                110            - -      Ser Glu Leu Ala Asp Arg Leu Arg - # Leu Ala Phe Val Pro Gly        Arg Leu                                                                                      115     - #            120     - #            125                 - -      Gly Glu Thr Val Ala Ala Ala Leu - # Thr Thr Ser Phe Ser Ala       Phe Leu                                                                                  130         - #        135         - #        140                     - -      Ser Ala Leu Ser Gly His Pro Arg - # Leu Pro Gln Asn Ala Leu       Asn Ala                                                                              145             - #    150             - #    155             - #        160                                                                           - -      Leu Asn Ala Leu Thr Thr Asp Gly - # Val Ala Ala Phe Asn Arg        Gln Tyr                                                                                           - #   165              - #   170              - #         175                                                                              - -      Pro Gln Gly Leu Pro Asp Arg Trp - # Gly Gly Met Gly Pro Ala       Gln Val                                                                                          180 - #                185 - #                190            - -      Asn Ala Val His Tyr Tyr Ser Trp - # Ser Gly Ile Ile Lys Gly        Ser Arg                                                                                      195     - #            200     - #            205                 - -      Leu Ala Glu Ser Leu Asn Leu Leu - # Asp Pro Leu His Asn Ala       Leu Arg                                                                                  210         - #        215         - #        220                     - -      Val Phe Asp Ser Phe Phe Thr Arg - # Glu Thr Arg Glu Asn Asp       Gly Met                                                                              225             - #    230             - #    235             - #        240                                                                           - -      Val Gly Arg Phe Ser Ser His Leu - # Gly Gln Val Ile Arg Ser        Asp Tyr                                                                                           - #   245              - #   250              - #         255                                                                              - -      Pro Leu Asp His Leu Asp Thr Ile - # Asn His Met Ala Arg Gly       Ser Ala                                                                                          260 - #                265 - #                270            - -      Gly Ala Ser Thr Arg                                                              275                                                               - -  - - (2) INFORMATION FOR SEQ ID NO: 4:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 269 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO: 4:                       - -      Ser Ile Asp Gly Gly Ile Arg Ala - # Ala Thr Ser Gln Glu Ile        Asn Glu                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Leu Thr Tyr Tyr Thr Thr Leu Ser - # Ala Asn Ser Tyr Cys Arg       Thr Val                                                                                          20 - #                 25 - #                 30             - -      Ile Pro Gly Ala Thr Trp Asp Cys - # Ile His Cys Asp Ala Thr        Glu Asp                                                                                      35     - #             40     - #             45                  - -      Leu Lys Ile Ile Lys Thr Trp Ser - # Thr Leu Ile Tyr Asp Thr       Asn Ala                                                                                  50         - #         55         - #         60                      - -      Met Val Ala Arg Gly Asp Ser Glu - # Lys Thr Ile Tyr Ile Val       Phe Arg                                                                              65             - #     70             - #     75             - #         80                                                                            - -      Gly Ser Ser Ser Ile Arg Asn Trp - # Ile Ala Asp Leu Thr Phe        Val Pro                                                                                           - #   85               - #   90               - #         95                                                                               - -      Val Ser Tyr Pro Pro Val Ser Gly - # Thr Lys Val His Lys Gly       Phe Leu                                                                                          100 - #                105 - #                110            - -      Asp Ser Tyr Gly Glu Val Gln Asn - # Glu Leu Val Ala Thr Val        Leu Asp                                                                                      115     - #            120     - #            125                 - -      Gln Phe Lys Gln Tyr Pro Ser Tyr - # Lys Val Ala Val Thr Gly       His Ser                                                                                  130         - #        135         - #        140                     - -      Leu Gly Gly Ala Thr Ala Leu Leu - # Cys Ala Leu Gly Leu Tyr       Gln Arg                                                                              145             - #    150             - #    155             - #        160                                                                           - -      Glu Glu Gly Leu Ser Ser Ser Asn - # Leu Phe Leu Tyr Thr Gln        Gly Gln                                                                                           - #   165              - #   170              - #         175                                                                              - -      Pro Arg Val Gly Asp Pro Ala Phe - # Ala Asn Tyr Val Val Ser       Thr Gly                                                                                          180 - #                185 - #                190            - -      Ile Pro Tyr Arg Arg Thr Val Asn - # Glu Arg Asp Ile Val Pro        His Leu                                                                                      195     - #            200     - #            205                 - -      Pro Pro Ala Ala Phe Gly Phe Leu - # His Ala Gly Glu Glu Tyr       Trp Ile                                                                                  210         - #        215         - #        220                     - -      Thr Asp Asn Ser Pro Glu Thr Val - # Gln Val Cys Thr Ser Asp       Leu Glu                                                                              225             - #    230             - #    235             - #        240                                                                           - -      Thr Ser Asp Cys Ser Asn Ser Ile - # Val Pro Phe Thr Ser Val        Leu Asp                                                                                           - #   245              - #   250              - #         255                                                                              - -      His Leu Ser Tyr Phe Gly Ile Asn - # Thr Gly Leu Cys Ser                             260 - #                265                                    - -  - - (2) INFORMATION FOR SEQ ID NO: 5:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 297 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO: 5:                       - -      Asp Asp Asn Leu Val Gly Gly Met - # Thr Leu Asp Leu Pro Ser        Asp Ala                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Pro Pro Ile Ser Leu Ser Ser Ser - # Thr Asn Ser Ala Ser Asp       Gly Gly                                                                                          20 - #                 25 - #                 30             - -      Lys Val Val Ala Ala Thr Thr Ala - # Gln Ile Gln Glu Phe Thr        Lys Tyr                                                                                      35     - #             40     - #             45                  - -      Ala Gly Ile Ala Ala Thr Ala Tyr - # Cys Arg Ser Val Val Pro       Gly Asn                                                                                  50         - #         55         - #         60                      - -      Lys Trp Asp Cys Val Gln Cys Gln - # Lys Trp Val Pro Asp Gly       Lys Ile                                                                              65             - #     70             - #     75             - #         80                                                                            - -      Ile Thr Thr Phe Thr Ser Leu Leu - # Ser Asp Thr Asn Gly Tyr        Val Leu                                                                                           - #   85               - #   90               - #         95                                                                               - -      Arg Ser Asp Lys Gln Lys Thr Ile - # Tyr Leu Val Phe Arg Gly       Thr Asn                                                                                          100 - #                105 - #                110            - -      Ser Phe Arg Ser Ala Ile Thr Asp - # Ile Val Phe Asn Phe Ser        Asp Tyr                                                                                      115     - #            120     - #            125                 - -      Lys Pro Val Lys Gly Ala Lys Val - # His Ala Gly Phe Leu Ser       Ser Tyr                                                                                  130         - #        135         - #        140                     - -      Glu Gln Val Val Asn Asp Tyr Phe - # Pro Val Val Gln Glu Gln       Leu Thr                                                                              145             - #    150             - #    155             - #        160                                                                           - -      Ala His Pro Thr Tyr Lys Val Ile - # Val Thr Gly His Ser Leu        Gly Gly                                                                                           - #   165              - #   170              - #         175                                                                              - -      Ala Gln Ala Leu Leu Ala Gly Met - # Asp Leu Tyr Gln Arg Glu       Pro Arg                                                                                          180 - #                185 - #                190            - -      Leu Ser Pro Lys Asn Leu Ser Ile - # Phe Thr Val Gly Gly Pro        Arg Val                                                                                      195     - #            200     - #            205                 - -      Gly Asn Pro Thr Phe Ala Tyr Tyr - # Val Glu Ser Thr Gly Ile       Pro Phe                                                                                  210         - #        215         - #        220                     - -      Gln Arg Thr Val His Lys Arg Asp - # Ile Val Pro His Val Pro       Pro Gln                                                                              225             - #    230             - #    235             - #        240                                                                           - -      Ser Phe Gly Phe Leu His Pro Gly - # Val Glu Ser Trp Ile Lys        Ser Gly                                                                                           - #   245              - #   250              - #         255                                                                              - -      Thr Ser Asn Val Gln Ile Cys Thr - # Ser Glu Ile Glu Thr Lys       Asp Cys                                                                                          260 - #                265 - #                270            - -      Ser Asn Ser Ile Val Pro Phe Thr - # Ser Ile Leu Asp His Leu        Ser Tyr                                                                                      275     - #            280     - #            285                 - -      Phe Asp Ile Asn Glu Gly Ser Cys - # Leu                                      290         - #        295                                            - -  - - (2) INFORMATION FOR SEQ ID NO: 6:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 44 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO: 6:                       - - GCTGCTCTGC CTAACCCTTA CGACGACCCT TTCTACACCA CCCC   - #                      - # 44                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO: 7:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 80 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO: 7:                       - - TTCTTGAGCG CGCGGATGCC GTCGAGGATA GCCATGCCCT CTTCGTAGCC AG -             #CGATCCAG     60                                                                 - - GCGGCTTTGA AGCCTTCGTG            - #                  - #                      - # 80                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO: 8:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO: 8:                       - - CAGAACGAGG CGGTGGCCGA C           - #                  - #                      - #21                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 9:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 80 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO: 9:                       - - TTCTTGAGCG CGCGGATGCC GTCGAGGATA GCCATGCCCT CTTCGTAGCC AG -             #CGATGAAG     60                                                                 - - GCGGCTTTCC AGCCTTCGTG            - #                  - #                      - # 80                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO: 10:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO: 10:                      - - AACCCATACG ACGACCC             - #                  - #                      - #   17                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 11:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 80 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO: 11:                      - - TTCTTGAGCG CGCGGATGCC GTCGAGGATA GCCATGCCCT CTTCGTAGCC AG -             #CGATCCAG     60                                                                 - - GCGGCTTTCC AGCCTTCGTG            - #                  - #                      - # 80                                                                __________________________________________________________________________

We claim:
 1. A lipase variant comprising a modification in an amino acidsequence of a parent lipase having the amino acid sequence of SEQ IDNO:2, wherein the modification is the substitution of phenylalanine atposition 139 of the mature lipase (position 170 of SEQ ID NO:2) withtryptophan.
 2. A lipase variant comprising a modification in an aminoacid sequence of a parent lipase having the amino acid sequence of SEQID NO:2, wherein the modification is the substitution of phenylalanineat positions 135 and 139 of the mature lipase (positions 166 and 170 ofSEQ ID NO:2) with tryptophan.
 3. A lipase variant comprising amodification in an amino acid sequence of a parent lipase having theamino acid sequence of SEQ ID NO:3, wherein the modification is thesubstitution of phenylalanine at position 29 with trypophan.
 4. A lipasevariant comprising a modification in an amino acid sequence of a parentlipase having the amino acid sequence of SEQ ID NO:5, wherein themodification is the substitution of alanine at position 117 withtryptophan.
 5. A DNA construct comprising a DNA sequence encoding alipase variant according to claim
 1. 6. A recombinant expression vectorwhich carries a DNA construct according to claim
 5. 7. A host cell whichis transformed with a DNA construct according to claim
 5. 8. A method ofproducing a lipase variant, comprising(a) culturing a host cellaccording to claim 7 under conditions conducive to the production of thelipase variant and (b) recovering the lipase variant from the culture.9. An enzymatic process, comprising(a) reacting an ester with water; (b)reacting an acid with an alcohol; or (c) reacting an ester with an acid,an alcohol or a second ester, wherein the reaction is catalyzed by alipase variant according to claim
 1. 10. A process for hydrolyzing resinin pulp, comprising hydrolyzing the pulp with a lipase variant accordingto claim
 1. 11. A DNA construct comprising a DNA sequence encoding alipase variant according to claim
 2. 12. A recombinant expression vectorwhich carries a DNA construct according to claim
 11. 13. A host cellwhich is transformed with a DNA construct according to claim
 11. 14. Amethod of producing a lipase variant, comprising(a) culturing a hostcell according to claim 13 under conditions conducive to the productionof the lipase variant and (b) recovering the lipase variant from theculture.
 15. An enzymatic process, comprising(a) reacting an ester withwater; (b) reacting an acid with an alcohol; or (c) reacting an esterwith an acid, an alcohol or a second ester, wherein the reaction iscatalyzed by a lipase variant according to claim
 2. 16. A process forhydrolyzing resin in pulp, comprising hydrolyzing the pulp with a lipasevariant according to claim
 2. 17. A DNA construct comprising a DNAsequence encoding a lipase variant according to claim
 3. 18. Arecombinant expression vector which carries a DNA construct according toclaim
 17. 19. A host cell which is transformed with a DNA constructaccording to claim
 17. 20. A method of producing a lipase variant,comprising(a) culturing a host cell according to claim 19 underconditions conducive to the production of the lipase variant and (b)recovering the lipase variant from the culture.
 21. An enzymaticprocess, comprising(a) reacting an ester with water; (b) reacting anacid with an alcohol; or (c) reacting an eater with an acid, an alcoholor a second ester, wherein the reaction is catalyzed by a lipase variantaccording to claim
 3. 22. A process for hydrolyzing resin in pulp,comprising hydrolyzing the pulp with a lipase variant according to claim3.
 23. A DNA construct comprising a DNA sequence encoding a lipasevariant according to claim
 4. 24. A recombinant expression vector whichcarries a DNA construct according to claim
 23. 25. A host cell which istransformed with a DNA construct according to claim
 23. 26. A method ofproducing a lipase variant, comprising(a) culturing a host cellaccording to claim 25 under conditions conducive to the production ofthe lipase variant and (b) recovering the lipase variant from theculture.
 27. An enzymatic process, comprising(a) reacting an ester withwater; (b) reacting an acid with an alcohol; or (c) reacting an esterwith an acid, an alcohol or a second ester, wherein the reaction iscatalyzed by a lipase variant according to claim
 4. 28. A process forhydrolyzing resin in pulp, comprising hydrolyzing the pulp with a lipasevariant according to claim 4.